Arch Dermatol Res (2005) 297: 43–47 DOI 10.1007/s00403-005-0574-8
SH O RT CO MM U N IC A T IO N
MT Cruz Æ M Gonc¸alo Æ A Paiva Æ JM Morgado A Figueiredo Æ CB Duarte Æ MC Lopes
Contact sensitizers downregulate the expression of the chemokine receptors CCR6 and CXCR4 in a skin dendritic cell line
Received: 17 January 2005 / Revised: 12 April 2005 / Accepted: 19 April 2005 / Published online: 28 May 2005 Springer-Verlag 2005
Abstract Chemokines are involved in the control of dendritic cell (DC) trafficking, which is critical for the immune response, namely in allergic contact dermatitis (ACD). In this work, we investigated by flow cytometry the effect of the contact sensitizers 2,4-dinitrofluorobenzene (DNFB), 1,4-phenylenediamine (PPD) and nickel sulfate (NiSO4), on the surface expression of the chemokine receptors CCR6 and CXCR4 in DC. As an experimental model of a DC we used a fetal skin-derived dendritic cell line (FSDC), which has morphological, phenotypical and functional characteristics of skin DC. Our results show that all the skin sensitizers studied decreased the membrane expression of the chemokine receptors CCR6 and CXCR4. In contrast, 2,4-dichloronitrobenzene (DCNB), the inactive analogue of DNFB without contact sensitizing properties, was without effect on the surface expression of these receptors. Lipopolysaccharide (LPS), which induces the maturation of DC, also reduced surface CCR6 and CXCR4 expression. Keywords Skin dendritic cell Æ Contact sensitizers Æ CCR6 Æ CXCR4 Æ Nickel sulfate Æ DNFB Æ PPD Abbreviations ACD: Allergic contact dermatitis Æ DC: Dendritic cell Æ DCNB: 2,4Dichloronitrobenzene Æ DMSO: Dimethyl M. Cruz Æ M. Lopes (&) Rua do Norte, Faculdade de Farma´cia, Universidade de Coimbra, Coimbra, Portugal E-mail: mcfl
[email protected] Tel.: +351-239-480237 Fax: +351-239-480217 M. Cruz Æ C. Duarte Æ M. Lopes Centro de Neurocieˆncias e Biologia Celular, Universidade de Coimbra, Coimbra, Portugal M. Gonc¸alo Æ A. Figueiredo Faculdade de Medicina (Servic¸o de Dermatologia), Hospital da Universidade de Coimbra, Coimbra, Portugal A. Paiva Æ J. Morgado Centro de Histocompatibilidade do Centro, Coimbra, Portugal
sulphoxide Æ DNFB: 2,4-Dinitrofluorobenzene Æ FSDC: Fetal skin dendritic cell line Æ LC: Langerhans cells Æ LPS: Lipopolysaccharide Æ MFI: Mean fluorescence intensity Æ MTT: 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide Æ PPD: 1,4-Phenylenediamine
Introduction Epidermal skin dendritic cells (DC), namely Langerhans cells, play an important role in allergic contact dermatitis (ACD) and other cell-mediated immune reactions. They constantly monitor the epidermal microenvironment by taking up and processing antigens that can be recognized, after migration to the lymph nodes, by T cells [1]. The migration of DC, both to sites of inflammation and to the draining lymph nodes, is dependent on a switch in the expression of chemokine receptors and in the production of chemokines [2]. Chemokines constitute a family of structurally related small (67–127 amino acids, 8–14 kDa) chemotactic cytokines that regulate the migration of leucocytes throughout the body, both under physiological and inflammatory conditions. They are the only cytokines that act on the superfamily of G-protein-coupled serpentine receptors [3], namely class A, which are characterized by high homology with rhodopsin, the prototypical family member [4]. In the skin, the migration and epidermal retention of Langerhans cells is dependent on its expression of CCR6, which is downregulated as DC mature and migrate to the lymph nodes. CXCR4, the membrane chemokine receptor for CXCL12, has been related with the constitutive basal trafficking of leucocytes, namely DC [2]. Scarce information is available concerning direct effects of contact sensitizers on chemokine receptor expression in skin DC. Therefore, the aim of this study was to investigate whether different contact sensitizers, namely 2,4-dinitrofluorobenzene (DNFB), 1,4-pheny-
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lenediamine (PPD) and nickel sulfate (NiSO4), modulate the surface expression of the chemokine receptors CCR6 and CXCR4 in DC. As an experimental model of a DC we used a fetal skin-derived dendritic cell line (FSDC), which has morphological, phenotypical and functional characteristics of a skin DC, the Langerhans cells [5]. As a positive control we used lipopolysaccharide (LPS), which was previously shown to induce FSDC activation [6, 7] and DC maturation [8]. As a negative control we used 2,4-dichloronitrobenzene (DCNB), the inactive analogue of DNFB which does not induce ACD.
MTT assay Assessment of MTT reduction by metabolically active cells was made by a colorimetric assay, using 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) as previously reported [6]. In four independent experiments, the average cell viability for FSDC treated, for 18 hours, with the refereed concentrations of nickel and PPD was above 90%, and for DNFB it was respectively 76% and 64%, for 1 lg/ml and 2.5 lg/ml (data not shown). DCNB and the vehicle DMSO had no effect on cell viability evaluated by the MTT assay (data not shown).
Materials and methods Materials The PE-conjugated monoclonal antibodies against CCR6 and CXCR4 were purchased from R&D Systems (Lille, France). DNFB and DCNB were from SigmaAldrich Quı´ mica (Madrid, Spain) and PPD was from Aldrich Chemical Co. (Milwaukee, WI). LPS from Escherichia coli (serotype 026:B6) was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Fetal calf serum was from Biochrom KG (Berlin, Germany) and trypsin from Invitrogen (Paisley, UK). All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA).
Flow cytometric analysis Briefly, FSDC cells treated with culture medium alone (control), or with the above refereed chemicals (concentrations indicated in the Figs. 1 and 2) were washed with phosphate buffered saline, and cell pellets were incubated with PE-conjugated monoclonal antibodies against CCR6 or CXCR4, for 10 min at room temperature. After washing again with saline, flow cytometry analysis was performed with FACScalibur flow cytometer and CellQuest software (Becton Dickinson). Appropriate isotype controls used at the same concentration as the test antibody showed a mean fluorescence
Cell culture and chemicals A fetal mouse skin dendritic cell line (FSDC), kindly supplied by Dr. G. Girolomoni, was used in the experiments. This cell line has a surface phenotype consistent with that of Langerhans cell progenitor (H-2d.b+, I-Ad.b+, CD54+, MHCII+, MHCI+, CD11c+, CD11b+, B7.2+, CD44+ B220 , CD3 ). After treatment with cytokines, FSDC stimulate allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction and present haptens to primed T cells in vitro. Moreover, FSDC derivatized with haptens and injected either intravenously or subcutaneously efficiently induce contact sensitivity responses in naı¨ ve syngeneic mice [5]. Cells were cultured in endotoxin-free Iscove’s medium supplemented with 10% (v/v) fetal calf serum, 1% (w/v) glutamine, 3.02 g/l sodium bicarbonate, 100 lg/ml streptomycin and 100 U/ml penicillin. For flow cytometry analysis and MTT assay, FSDC were plated at 2·106 cells/well, in 6-well culture plates, or at 0.2·106 cells/well, in 48-well culture plates, respectively, and then stimulated, for 18 h, with culture medium alone, or with LPS (2 lg/ml), or with the following contact sensitizers: PPD (10, 50 lg/ml) and NiSO4 (50 lg/ml), dissolved in saline, DNFB (1, 2.5 lg/ml) and DCNB (2.5 lg/ml) pre-solubilized in dimethyl sulphoxide (DMSO) and diluted in saline. DMSO concentration never exceeded 0.025% in the culture medium.
Fig. 1 Effect of contact sensitizers and LPS on CCR6 expression in FSDC cells. FSDC cells (2·106 cells) were incubated in culture medium alone (control), or with LPS, or in the presence of different contact sensitizers, at the indicated concentrations, for 18 h. Surface expression of CCR6 was analyzed by flow cytometry as described in material and methods and data are expressed as percentage of non-treated control cells (100% corresponds to a mean fluorescence intensity of 29.6±3.5). Each value represents the mean±SEM from the indicated number of experiments (*P
